The mutagenic and carcinogenic effects of electrophiles such as N-methyl-N-nitrosourea are mainly due to the O6-alkylation of guanine in DNA. To protect themselves against DNA-alkylation, mammals and bacteria possess a protein, O6-alkylguanine-DNA alkyltransferase (AGT) which repairs these lesions. AGT transfers the alkyl group from the position O6 of alkylated guanine and guanine derivatives to the mercapto group of one of its own cysteines, resulting in an irreversibly alkylated AGT. The underlying mechanism is a nucleophilic reaction of the SN2 type which explains why not only methyl groups, but also benzylic groups are easily transferred. As overexpression of AGT in tumour cells is the main reason for resistance to alkylating drugs such as procarbazine, dacarbazine, temozolomide and bis-2-chloroethyl-N-nitrosourea, inhibitors of AGT have been proposed for use as sensitisers in chemotherapy (Pegg et al., Prog Nucleic Acid Res Mol Biol 51:167-223, 1995). WO 97/20843 describes 4-heteroarylmethoxy-pyrimidines acting as AGT depleting compounds and their use for depleting AGT levels in tumor cells and thereby increasing responsiveness to alkylating anti-tumor drugs.
WO 02/083937 discloses a method for detecting and/or manipulating a protein of interest wherein the protein is fused to AGT and the AGT fusion protein contacted with an AGT substrate carrying a label, and the AGT fusion protein detected and optionally further manipulated using the label. Several AGT fusion proteins to be used, general structural principles of the AGT substrate and a broad variety of labels and methods to detect the label useful in the method are described.
WO 2004/031404 describes particular AGT fusion proteins to be used in the mentioned method for detecting and/or manipulating a protein of interest, labelled fusion proteins obtainable by this method, and the method using the particular AGT fusion proteins; and the related WO 2004/031405 discloses additional AGT substrates carrying a label particularly suitable in the mentioned method for detecting and/or manipulating a protein of interest, and the application of such particularly labelled substrates.
Unpublished PCT/EP2005/050899 describes AGT mutants suitable in the method. Related unpublished PCT/EP2005/050900 discloses further AGT substrates with a guanine (purine), azapurine or pyrimidine nucleus carrying a label particularly suitable in the mentioned method for detecting and/or manipulating a protein of interest, and the application of such particularly labelled substrates.